KR101607783B1 - Antibodies to pcsk9 and uses thereof - Google Patents

Abstract

The present invention relates to a composition comprising an antibody or antigen-binding fragment thereof, such a PCSK9 antibody or antigen-binding fragment, and a composition for prophylactic or therapeutic treatment of hyperlipidemia or hypercholesterolemia, And a method of using the same.

Description

ANTIBODIES TO PCSK9 AND USES THEREOF < RTI ID = 0.0 >

The present invention relates to the field of pharmaceuticals. Specifically, the present invention relates to antibodies against the full-length protein converter subtilisin / quicksch type 9 (PCSK9), compositions comprising such PCSK9 antibodies, and methods of using PCSK9 antibodies in the treatment of hyperlipidemia or hypercholesterolemia .

The prodrug protein concentrator subtilisin / ephedrine type 9 (PCSK9) is a secreted serine protease mainly produced in the liver and regulates the plasma concentration of low density lipoprotein cholesterol (LDL-C). Secreted PCSK9 binds to and is internalized by the LDL receptor (LDLR) located on the surface of the hepatocytes. LDLR functions to cleave LDL-C from plasma by binding to LDL particles and transporting them to lysosomes for degradation. Once LDL particles are delivered for degradation, LDLR recirculates to the surface of the hepatocyte cell and binds to additional LDL-C, clearing them from the plasma. PCSK9 regulates plasma LDL-C by directing the internalized LDLR to degradation rather than recycling to the cell surface, thereby reducing LDL-C clearance. Studies in rodents with defective or overexpressed PCSK9 have now confirmed that PCSK9 regulates circulating LDL levels by regulating the level of LDLR. The observation that circulating PCSK9 is involved in the degradation of hepatic LDLR suggests that antibody neutralization of PCSK9 is an operative treatment for LDL-C depression. It has also been reported that statin drugs, the current standard therapy for the lowering of LDL-C, can actually increase PCSK9 expression and serum levels. Thus, PCSK9 antibodies are also likely to reduce LDL-C in a synergistic manner with statin therapy.

Their effects on PCSK9 antibodies and plasma LDL-C depression are known in the art. For example, US2009 / 0246192, US2009 / 0142352, US2010 / 0166768 and WO2010 / 029513 disclose such PCSK9 antibodies and their use. However, to date, no antibody that targets PCSK9 has been approved for therapeutic use. Thus, there is a need for an alternative PCSK9 antibody. In particular, there remains a need for an alternative PCSK9 antibody that reduces LDL-C to high efficacy. More specifically still, there is a need for an alternative PCSK9 antibody that reduces LDL-C to high efficacy and provides a sustained duration of action (e.g., sustained inhibition of LDL-C levels). Such antibodies will preferably also possess good physicochemical properties to facilitate the development, manufacture or formulation.

Wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein the HCVR comprises the complementarity determining regions The amino acid sequence of HCDR1 is represented by SEQ ID NO: 1, the amino acid sequence of HCDR2 is represented by SEQ ID NO: 2, the amino acid sequence of HCDR3 is represented by SEQ ID NO: 3, the amino acid sequence of LCDR1 is represented by SEQ ID NO: 4, 5, and wherein the amino acid sequence of LCDR3 is represented by SEQ ID NO: 6. In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to human PCSK9. (HCVR) and a light chain variable region (LCVR), wherein the amino acid sequence of the HCVR is represented by SEQ ID NO: 7, and the amino acid sequence of the LCVR is represented by SEQ ID NO: 8. In one embodiment, the present invention includes a heavy chain variable region Lt; RTI ID = 0.0 > PCSK9 < / RTI > or antigen-binding fragment thereof. In another embodiment, the invention comprises two heavy chain variable regions (HCVRs) and two light chain variable regions, wherein the amino acid sequence of each HCVR is represented by SEQ ID NO: 7, and the amino acid sequence of each LCVR is represented by SEQ ID NO: 8 Or an antigen-binding fragment thereof.

In another specific embodiment, the invention encompasses an antibody that comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is represented by SEQ ID NO: 9, the amino acid sequence of LC is represented by SEQ ID NO: 10, Antigen-binding fragment. In another specific embodiment, the invention comprises two heavy chains (HC) and two light chains (LC), wherein the amino acid sequence of each HC is represented by SEQ ID NO: 9 and the amino acid sequence of each LC is represented by SEQ ID NO: 10 ≪ / RTI > In a most particular embodiment, the invention provides an antibody consisting of two HCs and two LCs, wherein the amino acid sequence of each HC is represented by SEQ ID NO: 9, and the amino acid sequence of each LC is represented by SEQ ID NO: 10 .

The invention also provides a pharmaceutical composition comprising an antibody or antigen-binding fragment of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient. More specifically, the pharmaceutical compositions of the present invention further comprise one or more additional therapeutic agents.

The present invention also provides a method of treating hyperlipidemia or hypercholesterolemia, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment of the invention. The invention also provides an antibody or antigen-binding fragment thereof of the invention for use in therapy. More particularly, the present invention provides an antibody or antigen-binding fragment thereof of the invention for use in the treatment of hyperlipidemia or hypercholesterolemia. The invention also provides the use of the antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for the treatment of hyperlipidemia or hypercholesterolemia.

The invention also relates to nucleic acid molecules and expression vectors encoding the antibodies or antigen-binding fragments of the invention. (A) a host cell comprising a first polynucleotide sequence encoding a polypeptide sequence of SEQ ID NO: 9 and a second polynucleotide sequence encoding a second polypeptide sequence of SEQ ID NO: 10, Lt; RTI ID = 0.0 >expression; < / RTI > And (b) recovering an antibody comprising a heavy chain and a light chain from the host cell, wherein the polypeptide sequence of the heavy chain is represented by SEQ ID NO: 9 and the polypeptide sequence of the light chain is represented by SEQ ID NO: 10 ≪ / RTI > More specifically, the antibody produced by the above method comprises two heavy chains and two light chains, wherein the polypeptide sequence of each heavy chain is represented by SEQ ID NO: 9, and the polypeptide sequence of each light chain is represented by SEQ ID NO: 10.

A full-length antibody is an immunoglobulin molecule comprising two heavy (H) chains and two light (L) chains linked by a disulfide bond. The amino terminus of each chain includes a variable region of about 100-110 amino acids primarily responsible for antigen recognition through the complementarity determining region (CDR) contained therein. The carboxy terminus of each chain defines a constant region primarily responsible for the effector function. Light chains are classified as kappa or lambda, each of which is characterized by a specific constant region as known in the art. The heavy chain is classified as gamma, mu, alpha, delta or epsilon and defines the isotype of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be subdivided into subclasses, such as IgG1, IgG2, IgG3, and IgG4. Each heavy chain type is also characterized by a specific constant region having a sequence known in the art. As used herein, an "antigen-binding fragment" refers to a Fab fragment, Fab 'fragment, F (ab') ₂ fragment, and single chain Fv fragment that bind to human PCSK9. As used herein, the term "binding to human PCSK9" refers to the interaction with an epitope on human PCSK9 expressed in the amino acid sequence of SEQ ID NO: 14. The term "epitope" as used herein refers to a discontinuous three-dimensional site on the antigen recognized by the antibody or antigen-binding fragment of the invention.

CDRs are scattered in more conserved areas called framework areas ("FR"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs arranged in the amino terminal to carboxy terminal direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4. The three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3", and the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3". CDRs contain most of the residues that form specific interactions with the antigen. The numbering and positioning of CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragment of the present invention can be performed according to the well-known Kabat numbering rules (LCDR1-3, HCDR2-3) or Kabat + Can be determined according to Chothia (HCDR1).

Methods of producing and purifying antibodies and antigen-binding fragments are well known in the art and are described, for example, in Harlow and Lane (1988) Antibodies , A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15]. For example, a mouse can be immunized with human PCSK9 or a fragment thereof, followed by recovery and purification of the resulting antibody, followed by determination of the amino acid sequence using conventional methods known in the art. Antigen-binding fragments can also be prepared by conventional methods. An antibody or antigen-binding fragment of the invention is engineered to contain one or more human framework regions around a CDR from a non-human antibody. Human framework wiring sequences can be obtained from ImMunoGeneTics (IMGT) through their website http://imgt.cines.fr, or from The Immunoglobulin Facts Book by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351. Specific light-heavy frameworks for use in the antibodies or antigen-binding fragments of the invention include A3 and O2. Specific wired heavy chain framework regions for use in the antibody or antigen-binding fragment of the invention include VH3-21 and VH3-23.

The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified using known methods. For example, a cDNA sequence encoding a heavy chain (for example, the amino acid sequence shown by SEQ ID NO: 9) and a light chain (for example, the amino acid sequence shown by SEQ ID NO: 10) is cloned into a GS (glutamine synthetase) Can be operated. The engineered immunoglobulin expression vector can then be stably transfected into CHO cells. As will be appreciated by those skilled in the art, mammalian expression of the antibody will typically result in glycation at the highly conserved N-glycation site in the Fc region. Stable clones can be verified for the expression of antibodies that specifically bind to human PCSK9. Positive clones can be expanded in serum-free culture media for antibody production in a bioreactor. The medium in which the antibody is secreted can be purified by a conventional technique. For example, the medium can conveniently be applied to a Protein A or G Sepharose FF column equilibrated with a suitable buffer such as phosphate-buffered saline. The column is washed to remove non-specific binding components. The binding antibody is eluted, for example, with a pH gradient, and the antibody fraction is detected, for example, by SDS-PAGE, and then collected in one place. Antibodies can be enriched and / or sterile filtered using conventional techniques. Soluble aggregates and multimers can be effectively removed by conventional techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be frozen immediately, for example at -70 ° C, or lyophilized.

The antibody of the present invention is a monoclonal antibody. As used herein, "monoclonal antibody" or "mAb" refers to an antibody derived from, for example, a single copy or clone, including any eukaryote, prokaryote or phage clone. The monoclonal antibodies and antigen-binding fragments thereof can be used, for example, in hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR-grafting, or combinations of such techniques or other techniques known in the art . ≪ / RTI >

In another embodiment of the invention, the antibody or antigen-binding fragment thereof, or the nucleic acid encoding it, is provided in isolated form. As used herein, the term "isolated" refers to a protein, peptide, or nucleic acid that lacks or is substantially free of other polymeric species found in the cellular environment. &Quot; Substantially absent "as used herein means that the protein, peptide or nucleic acid of interest comprises more than 80% (by mole), preferably more than 90%, more preferably more than 95% .

Antibodies or antigen-binding fragments of the invention can be used in the treatment of patients. The term " treating "(or" treating "or" treating ") refers to slowing, stopping, stopping, alleviating, stopping, reducing or reversing the progression or severity of an existing symptom, disorder, condition or disease. As used herein, "patient" refers to a human or non-human mammal, preferably a human. As used herein, the term "effective amount " refers to the amount or dose of an antibody or antigen-binding fragment of the invention that provides a desired effect in a patient under treatment in single or multi-dose administration to a patient. An effective amount is a species of mammal; His size, age, and overall health; The particular disease involved; The degree or severity of the disease; Individual patient response; The particular antibody being administered; Mode of administration; Bioavailability characteristics of the formulation to be administered; Selected capacity prescription; And the use of any concomitant medicament, as will be readily apparent to those skilled in the art.

The antibody or antigen-binding fragment of the invention, or a pharmaceutical composition comprising it, can be administered by a parenteral route (e. G., Subcutaneous, intravenous, intraperitoneal, intramuscular or transdermal). The pharmaceutical compositions of the present invention may be prepared by methods known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 19 ^th ed. (1995), A. Gennaro et al., Mack Publishing Co.) and includes the antibody or antigen-binding fragment thereof disclosed herein, and one or more pharmaceutically acceptable carriers, diluents or excipients. For example, an antibody or antigen-binding fragment of the invention can be formulated with agents such as sodium citrate, citric acid, polysorbate 80, and sucrose, and the resulting composition is then lyophilized and dried at 2-8 [deg.] C Lt; / RTI > The lyophilized composition may then be reconstituted with the sterilized water for injection prior to administration.

The following examples illustrate the invention in more detail, but it is understood that the examples are given by way of illustration and not of limitation, and that various modifications may be made by those skilled in the art.

Example 1

Operation PCSK9  Antibody

The murine host was immunized with a peptide containing the C-terminal truncation fragment (SEQ ID NO: 17) of human PCSK9 and the PCSK9 binding IgG antibody was isolated and cloned using standard methods. The CDRs of the isolin murine Fabs were randomized by mutagenesis and the resulting antibodies were screened for affinity to human PCSK9. Affinity-promoting mutations were combined and optimized CDRs were engineered into human VH3-21 and A3 heavy and light chain frameworks, respectively. To further optimize the biophysical properties of humanized antibodies, targeted substitutions of aromatic and hydrophobic amino acids in the CDR sequence were performed. Randomized CDR libraries were also screened for additional affinity-enhancing mutations. The beneficial CDR mutations were expressed in random combination and the resulting antibodies were screened for affinity to human PCSK9. A full-length humanized-optimized PCSK9 antibody having the following amino acid sequence was obtained:

The corresponding cDNA sequences encoding the heavy and light chain amino acid sequences of SEQ ID NOS: 9 and 10, respectively, are as follows:

Example  2

Operation PCSK9  Expression of antibodies

The engineered PCSK9 antibody of Example 1 can be expressed in a stably transfected CHO cell line. (GS) expression vector containing the cDNA of SEQ ID NO: 11 (coding for the heavy chain amino acid sequence of SEQ ID NO: 9) and SEQ ID NO: 12 (coding for the light chain amino acid sequence of SEQ ID NO: 10) was used for electroporation to generate Chinese hamster cell line CHOK1SV Lonza Biologics PLC, Slough, UK). ≪ / RTI > The expression vector encodes the SV early (simian virus 40E) promoter and the gene for GS. Expression of GS enables the biochemical synthesis of glutamine, the amino acid required by CHOK1SV cells. After transfection, cells were bulk selected with 50 [mu] M L-methionine sulfoximine (MSX). The inhibition of GS by MSX was used to increase the rigidity of selection. Cells in which the expression vector cDNA is integrated into the transcriptional activation domain of the host cell genome can be screened against CHOK1SV wild type cells expressing endogenous GS. Bulk cultures were subjected to single-cell cloning using a fluorescence-activated cell sorting (FACS) technique and screened for the expression of the engineered PCSK9 antibody of Example 1 by expanding the clone cell line.

Example 3

Epitope  Combination

The PCSK9 binding epitope of murine IgG (from which the engineered PCSK9 antibody from Example 1 was derived) was determined by epitope extraction and hydrogen / deuterium exchange mass spectrometry and the linear amino acid sequence 160-181 of the catalytic domain of human PCSK9 (28 amino acid signal peptides Lt; RTI ID = 0.0 > PCSK9 < / RTI > The interaction between the working antibody of Example 1 and the relevant epitope in the catalytic domain of human PCSK9 was confirmed by evaluating its binding to synthetic peptides corresponding to residues 160-181 (Table 1). The working antibody of Example 1 binds to peptides 160-181 at a higher affinity than intact human PCSK9 and the difference is driven by a faster association rate (k _on ) _. The dissociation rate (k _off ) is within 2-fold of intact PCSK9, suggesting that the intensity of the interaction (after binding occurs) is similar. The data also suggests that almost all of the binding determinants are contained within the linear region of PCSK9. Binding of the working antibody of Example 1 to peptides 166-181 was significantly weaker than peptides 160-181, demonstrating the role of amino acids (or multiple amino acids) in the 160-165 region. The binding of the working antibody of Example 1 to peptides 163-174 was significantly stronger than that of 166-181, which also suggests a contribution from residues 163-165.

Example 4

Coupling dynamics and affinity

The binding kinetics and affinity of the test PCSK9 antibody against human, synomolgus monkey, mouse, rat, and rabbit PCSK9 were evaluated using the subject-known surface plasmon resonance (SPR) assay. Under physiological buffer conditions (ionic strength and pH) and temperature (37 캜), the working antibody of Example 1 had an average association rate (k _on ) of 1.2 x 10 ⁵ M ^-1 s ^-1 and And binds to human PCSK9 at an average dissociation rate (k _off ) of 1.2 x 10 < ^-3 > s < ^{-1 &} gt ;. The mean K _D for human PCSK9 binding for the working antibody of Example 1 was determined to be about 11 nM. Operation antibody of Example 1 is 1.1 x 10 ⁵ M ^-1 s ^-1 average speed association (k _on) and of The average dissociation rate (k _off ) of 2.5 x 10 < ^-3 > s < ^-1 > binds to synomolgus PCSK9 and a K _D for the synomolcus PCSK9 binding of about 25 nM is derived. Table 2 below summarizes the additional results obtained with the engineered PCSK9 antibodies of Example 1 using mouse, rat and rabbit PCSK9. These data indicate that the engineered PCSK9 antibody of Example 1 binds nano-affinity to both human and cynomolgus PCSK9 under physiological conditions of pH, ionic strength and temperature.

Example  5

LDL  For the receptor PCSK9  Inhibition of binding

PCSK9 regulates plasma LDL-C by reducing liver LDLR content and thereby decreasing LDL absorption by hepatocytes. The catalytic domain of PCSK9 is the site that binds to LDLR. Thus, antibodies recognizing the catalytic domain of PCSK9 are expected to inhibit the binding of PCSK9 to LDLR.

Using the alpha Li (AlphaLISA) ^® format to measure the effect of test PCSK9 antibody on PCSK9 binding to the LDL receptor. The recombinant full-length PCSK9 used in the assay was expressed as a C-terminal HIS-tagged protein in human embryonic kidney (HEK) 293 stable cell line (Qian et al., J. Lipid Res. 48: 1488-1498, 2007) . The recombinant LDL receptor extracellular domain was expressed as a C-terminal FLAG-tagged protein in transiently transfected HEK 293E cells (Qian et al., J. Lipid Res. 48: 1488-1498, 2007). Murine anti-PCSK9 Mab binding to the C-terminal domain of human PCSK9 was expressed in HEK293 cells and purified on a Protein-G affinity column followed by Superdex 200. Monoclonal anti-Flag ^® Bio M 2 (ANTI-FLAG ^® BioM2) antibody (Sigma, Sigma) is an antibody with purified mouse IgG1 monoclonal day is covalently attached to biotin by a hydrazide bond. The anti-flag bioM 2 will recognize the FLAG sequence at the N-terminus, Met-N-terminus or C-terminus of the FLAG fusion protein. The anti-flag bioM 2 can be detected by an avidin or streptavidin conjugate. Monoclonal anti-flags BioM ™ 2-biotin was supplied in 150 mM NaCl containing 50% glycerol, 10 mM sodium phosphate, pH 7.25, 0.02% sodium azide and stored at -20 ° C.

Alpha Liza ^® experiments were performed in 25 mM HEPES; well buffer (pH 7.0), pH 7.5, 100 mM NaCl, 2.5 mM CaCl ₂ , 0.5% TX-100, 0.1% casein, 1 mg / mL dextran- Proxy Plate (Perkin Elmer). Black was used as the alpha Manager ^® streptavidin donor beads (Perkin Elmer) and an alpha Li a murine anti -PCSK9 ^® Mab conjugated to acceptor beads. When the beads were brought close to each other through the interaction of the binding partners PCSK9 and LDLR, singlet oxygen was transferred from the donor bead to the acceptor bead. At laser excitation at 680 nm, singlet oxygen excited acceptor beads to emit light. The acceptor beads were bound to murine anti-PCSK9 Mab by reductive amination using NaBH ₃ CN (Sigma) and stored at 4 ° C. Murine anti-PCSK9 Mab-junction acceptor beads (22 [mu] g / mL) were preloaded with 2.22 nM PCSK9 for 1 hour. For an hour donor bead (44 ㎍ / mL) 5.55 nM anti-was loaded into pre-Flag ^® Bio M 2 and 2.22 nM FLAG- tagged LDLR. After preloading, 2 μl of the test PCSK9 antibody or control IgG was mixed with 9 μl of each bead mixture (final concentration of PCSK9 and LDLR = 1 nM) using a fully automatic Multimek (Beckman) Was added to the proxy plate and allowed to bind overnight at room temperature. Alpha Manager ^® signal (counts per second) was measured on a former in-Turbo (Envision Turbo) (Perkin Elmer). All experiments performed with the alpha riser ^® assay were performed under stellar conditions.

After the process carried out substantially as described above, a combination of human PCSK9 on administrator alpha LDLR in black ^® was increased as a function of the concentration of PCSK9. Addition of the working PCSK9 antibody of Example 1 (test PCSK9 antibody) resulted in a concentration-related and complete inhibition of PCSK9 binding to LDLR with an average IC ₅₀ of about 90 pM. Control IgG4 was ineffective in the assay. The results of this assay demonstrated that the engineered PCSK9 antibody of Example 1 inhibited the binding of human PCSK9 to LDLR.

Example 6

HepG2  For cells PCSK9  Suppress Function

Test on Density of Hepatocyte LDLR To determine the effect of the PCSK9 antibody, human HepG2 cells were cultured in a poly-D-lysine coated T75 flask. After 24 hours, cells were resuspended in 5% (v / v) human lipoprotein-depleted serum (LPDS; Intracel) in poly-D-lysine coated 96-well black plates (Becton-Dickinson) At 5,000 cells / well in 100 [mu] L of DMEM / F-12 (3: 1) medium. After overnight incubation in the LPDS-containing medium, the cells were incubated for 2 hours at a concentration ranging from 2.6 to 1333 nM with 69 nM (5 [mu] g / mL) C-terminal HIS-tagged recombinant human PCSK9 and test PCSK9 antibody or IgG4 control antibody . All incubations were performed at 37 < 0 > C. LDLR levels were monitored as a xenon fluoride Alexa ^® 488 ^® mouse IgG2b labeling kit ^{(Zenon ® Alexa Fluor ® 488 Mouse} IgG2b Labeling Kit) ( Invitrogen) with fluorescence-labeled antibodies LDLR (peurojen (Progen)). The cells were incubated with the detection antibody for 90 min at room temperature and then fixed for 10 min using a formalin-free fixative (ANTECH, Ltd., Prefer), followed by 0.01% triton X-100. The cells were stained with propidium iodide (Invitrogen) at 10 占 퐂 / mL and the total number of cells was measured. The LDLR signal was quantified using an Aquamen Explorer ^TM laser-scanning fluorescent microplate cytometer fluorescence detector (TTP LabTech).

Substantially following the procedure described above, human PCSK9 resulted in a concentration-dependent reduction of LDLR on HepG2 cells with an EC ₅₀ of 18 nM. The engineered PCSK9 antibody of Example 1 (test PCSK9 antibody) inhibited PCSK9-induced inhibition of LDLR on HepG2 cells with an IC ₅₀ of 104 nM. The human IgG4 control was relatively inactive at concentrations up to 1333 nM. These data demonstrated that the engineered PCSK9 antibody of Example 1 inhibits PCSK9-mediated LDLR degradation.

Example 7

LDL  Absorption PCSK9 - Suppression of induced decline

Test on the Absorption of LDL To determine the effect of the PCSK9 antibody, HepG2 cells were seeded in 100 μl DMEM / F-12 (3: 1) medium supplemented with 5% human LPDS on poly-D-lysine coated 96- to under 5,000 cells / well are seeded and 5% CO ₂ atmosphere at 37 ℃ was incubated for 18 hours. Human PCSK9 (69 nM) was added to the cells in the presence or absence of the PCSK9 test antibody or human IgG4 control at a concentration ranging from 2.6 nM to 1333 nM and pre-incubated with the cells for 2 hours at 37 < 0 > C. After addition of 100 ng / well of fluorescent-labeled LDL (BODIPY-LDL, Invitrogen), the cells were then incubated for 4 hours at 37 ° C. The cells were fixed at room temperature for 20 min on a formalin-free fixative (Frefer; Anertec Limited). After washing the cells twice with PBS, the cells were permeabilized with PBS buffer containing 0.01% Triton X-100 at room temperature for 15 min, and then stained with propidium iodide at 10 / / mL to measure the total number of cells. LDL absorption was measured as% of fluorescent cells for total cells as measured using an Aquaman Explorer ^TM laser-scanning fluorescence microplate cytometer. The response to the test PCSK9 antibody or control IgG was expressed as% inhibition of PCSK9, i.e. as a% return to maximum LDL uptake in the absence of PCSK9 versus baseline LDL-C uptake in the presence of PCSK9 alone. Corresponding IC ₅₀ values for inhibition of PCSK9-induced reduction of LDL absorption were also calculated.

Substantially following the procedure described above, human PCSK9 resulted in a concentration-related decrease in LDL absorption in HepG2 cells with an EC ₅₀ of 32 nM. The engineered PCSK9 antibody of Example 1 reversed PCSK9-induced suppression, which was reflected in increased LDL uptake, whereas control IgG did not reverse the inhibition. Specifically, the operation PCSK9 antibody of Example 1 exhibited 84% of the PCSK9 average maximum% inhibition and 194 nM average IC ₅₀ of the. These data demonstrated that the engineered PCSK9 antibody of Example 1 suppresses PCSK9-induced reduction of LDL absorption.

Example  8

In vivo  efficacy

To determine the in vivo pharmacokinetics (PK) and / or pharmacodynamics (PD) effects of the test PCSK9 antibody, the test antibody can be administered to a normal sinomorphous monkey and subsequently various PK and / or PD parameters can be measured have. For example, the test PCSK9 antibody can be administered intravenously or subcutaneously to a healthy naive sinomorphous monkey and the serum concentration of the test antibody can then be measured using a human IgG sandwich ELISA. After antibody administration can be by using a serum concentration taken over the various time points to measure the various PK parameters for the test antibody, including the _{T 1/2, C max,} AUC and plasma elimination (CL). Similarly, the test can be administered intravenously or subcutaneously PCSK9 Antibody in Healthy naive Sino Mall Goose Monkey and LDL-C serum concentration of auto-analyzer (Direct eldi El-seeds plus Second Zhen (Direct LDL-C Plus, 2 nd Gen.), Roche Diagnostics).

Following substantially the procedure as described above, the pharmacokinetics of the engineered PCSK9 antibody of Example 1 were evaluated after single dose intravenous administration of 1, 5 or 15 mg / kg in healthy synomolgus monkeys and after single dose subcutaneous administration of 5 mg / kg After the dose was evaluated. The pharmacokinetic parameters measured from these studies are shown in Table 3 below.

Serum LDL was measured after administration of the modified PCSK9 antibody of Example 1 in two independent studies. In both studies, evidence of LDL-C inhibition was evident by post-24 hour post-administration with the engineered PCSK9 antibody of Example 1. A maximum mean LDL-C reduction of 60% (Study 1) and 25% (Study 2) was observed after intravenous (iv) administration of the antibody of Example 1 at 5 mg / kg. At 5 mg / kg i.v., mean LDL-C inhibition was maintained for approximately 8 weeks (Study 1) and 2 weeks (Study 2). In Study 2, there was a dose of moderate effect (1-15 mg / kg) on the size of LDL-C inhibition (25-35%). The effect of the dose on the duration of LDL-C inhibition was more apparent. Upon subcutaneous administration (5 mg / kg), the working antibody of Example 1 was effective in inhibiting LDL-C levels in a size similar to that observed after intravenous administration. There was no effect on serum high density lipoprotein cholesterol after administration of any dose of the working antibody of Example 1.

Example  9

Operation PCSK9  Physicochemical properties of antibodies

The engineered PCSK9 antibody of Example 1 was also found to have good solubility, chemical stability, and physical stability.

A. Solubility

Sufficient solubility is desired to allow for convenient administration. For example, a dose of 1 mg / kg administered in a 1.0 mL injection in a 100 kg patient will require a solubility of 100 mg / mL. It is also desirable to maintain the antibody in a monomeric state at high concentrations without high-molecular weight (HMW) aggregation. To determine the solubility of the test antibody, the antibody was incubated with (1) 10 mM citrate pH 6; (2) 10 mM citrate pH 6, 150 mM NaCl; And (3) dialysis into phosphate-buffered saline (PBS) pH 7.4. The recovered dialysate can then be analyzed by analytical size exclusion chromatography (SEC) to determine% HMW. The test antibody can then be concentrated at about 25 [deg.] C in a 4 mL centrifuge concentrator until the solubility limit is reached or the pore volume of the concentrator is reached. When the pore volume is reached, the concentration is reported as ≥. The% HMW can then be measured by analyzing the concentrated antibody with SEC. To determine if any increase in% HMW during concentration is reversible, a concentrated sample can be diluted to 1 mg / mL and analyzed with SEC.

Substantially following the procedure described above, the engineered PCSK9 antibody of Example 1 exhibited a solubility of greater than 128 mg / mL under all conditions tested. In addition, only low levels of HMW were present in high concentrations.

B. Chemical stability

Chemical stability promotes the development of formulations with sufficient shelf life. To assess the chemical stability of the test antibody, the antibody can be formulated at a concentration of 1 mg / mL in 10 mM citrate buffered at pH 4, pH 5, pH 6 or pH 7. The formulated samples were then incubated at 4 [deg.] C, 25 [deg.] C and 40 [deg.] C for 4 weeks with accelerated digestion studies. Changes in the charge profile of antibodies reflecting chemical changes can be assessed using capillary isoelectric focusing (cIEF) according to standard procedures. After performing the procedure substantially as described above, the chemical stability analysis of the working antibody of Example 1 provided the following results.

The results showed that after 4 weeks of storage at 25 DEG C, the percentile peak decreased by only 4.1 percentage points when formulated at pH 6 (the usual pH used for antibody preparations), indicating that the manipulated PCSK9 antibody of Example 1 was sufficient Suggesting that they have sufficient chemical stability to facilitate the development of solution formulations with a shelf life. Moreover, the antibody also exhibits good chemical stability at pH 5 and to a lesser extent at pH 7, suggesting that the antibody possesses stability characteristics that may allow formulation over a range of pH ranges.

C. Physical Stability

To assess the physical stability of the test antibody, the antibody was formulated at a protein concentration of 1 mg / mL in 10 mM citrate (or 10 mM Tris, pH 8) buffered at pH 4, pH 5, pH 6 or pH 7 . The samples were then incubated at 4 ° C, 25 ° C and 40 ° C for 4 weeks in an accelerated degradation study. After incubation, the physical stability was assessed using size exclusion chromatography (SEC) to separate the desired monomeric antibody from the aggregated high-molecular weight (HMW) antibody.

After performing the procedure substantially as described above, the physical stability analysis results of the engineered PCSK9 antibody of Example 1 are summarized in Table 6. The data show that at pH 5, pH 6 and pH 7, the change in HMW over 4 weeks at 25 ° C or 40 ° C was less than 1%, indicating that the antibody retains good physical stability and is capable of self- Suggesting resistance.

order

                         SEQUENCE LISTING <110> Eli Lilly and Company   <120> Antibodies to PCSK9 and Uses Thereof <130> X19477 <150> 61/535625 <151> 2011-09-16 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Gly Phe Pro Phe Ser Lys Leu Gly Met Val 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Synthetic <400> 2 Thr Ile Ser Gly Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly      <210> 3 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic <400> 3 Glu Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met Asp Tyr 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic <400> 4 Arg Ser Ser Lys Ser Leu Leu His Arg Asn Gly Ile Thr Tyr Ser Tyr 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial <220> <223> Synthetic <400> 5 Gln Leu Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic <400> 6 Tyr Gln Asn Leu Glu Leu Pro Leu Thr 1 5 <210> 7 <211> 125 <212> PRT <213> Artificial <220> <223> Synthetic <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Lys Leu             20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Thr Ile Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met             100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser         115 120 125 <210> 8 <211> 112 <212> PRT <213> Artificial <220> <223> Synthetic <400> 8 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Ser Ser Leu Leu His Arg             20 25 30 Asn Gly Ile Thr Tyr Ser Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Gln Leu Ser Asn Leu Ala Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Tyr Gln Asn                 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 9 <211> 451 <212> PRT <213> Artificial <220> <223> Synthetic <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Lys Leu             20 25 30 Gly Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Thr Ile Ser Gly Gly Gly Tyr Thr Tyr Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Glu Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met             100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr         115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser     130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His                 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser             180 185 190 Val Val Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys         195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu     210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Ser Glu Glu Glu Met Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Leu Gly     450 <210> 10 <211> 219 <212> PRT <213> Artificial <220> <223> Synthetic <400> 10 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Ser Ser Leu Leu His Arg             20 25 30 Asn Gly Ile Thr Tyr Ser Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Gln Leu Ser Asn Leu Ala Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Tyr Gln Asn                 85 90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu         115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe     130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu             180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser         195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 11 <211> 1356 <212> DNA <213> Artificial <220> <223> Synthetic <400> 11 gaggtgcagc tggtggagtc tgggggaggc ttggtaaagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cccgttcagt aagctcggca tggtttgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaacc attagtagtg gtggtggtta cacatactat 180 ccagacagtg tgaaggggcg gttcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc gagagaagga 300 attagctttc agggtggcac ctacacttat gttatggact actggggcca gggcaccctg 360 gtcaccgtct cctcagcctc caccaagggc ccatcggtct tcccgctagc gccctgctcc 420 aggagcacct ccgagagcac agccgccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600 ttgggcacga agacctacac ctgcaacgta gatcacaagc ccagcaacac caaggtggac 660 aagagagttg agtccaaata tggtccccca tgcccaccct gcccagcacc tgaggccgcc 720 gggggaccat cagtcttcct gttcccccca aaacccaagg acactctcat gatctcccgg 780 acccctgagg tcacgtgcgt ggtggtggac gtgagccagg aagaccccga ggtccagttc 840 aactggtacg tggatggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 900 ttcaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaac 960 ggcaaggagt acaagtgcaa ggtctccaac aaaggcctcc cgtcctccat cgagaaaacc 1020 atctccaaag ccaaagggca gccccgagag ccacaggtgt acaccctgcc cccatcccag 1080 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc 1140 gacatcgccg tggagtggga aagcaatggg cagccggaga acaactacaa gaccacgcct 1200 cccgtgctgg actccgacgg ctccttcttc ctctacagca ggctaaccgt ggacaagagc 1260 aggtggcagg aggggaatgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1320 tacacacaga agagcctctc cctgtctctg ggttga 1356 <210> 12 <211> 660 <212> DNA <213> Artificial <220> <223> Synthetic <400> 12 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtaa gagtctctta catcgtaatg gcatcactta ttcgtattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atcagctgtc caaccttgcc 180 tcaggagtcc cagacaggtt cagtggcagt gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggagtt tattactgct atcaaaatct agaacttccg 300 ctcacgttcg gccagggcac caaggtggaa atcaaacgga ctgtggctgc accatctgtc 360 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgctaa 660 <210> 13 <211> 672 <212> PRT <213> Artificial <220> <223> Synthetic <400> 13 Arg Ala Gln Glu Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala 1 5 10 15 Leu Arg Ser Glu Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr             20 25 30 Thr Ala Thr Phe His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly         35 40 45 Thr Tyr Val Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu     50 55 60 Arg Thr Ala Arg Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu 65 70 75 80 Thr Lys Ile Leu His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val                 85 90 95 Lys Met Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val             100 105 110 Asp Tyr Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp         115 120 125 Asn Leu Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln     130 135 140 Pro Pro Asp Gly Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser 145 150 155 160 Ile Gln Ser Asp His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp                 165 170 175 Phe Glu Asn Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala             180 185 190 Ser Lys Cys Asp Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly         195 200 205 Arg Asp Ala Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val     210 215 220 Leu Asn Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu 225 230 235 240 Glu Phe Ile Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val                 245 250 255 Val Leu Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala             260 265 270 Cys Gln Arg Leu Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly         275 280 285 Asn Phe Arg Asp Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu     290 295 300 Val Ile Thr Val Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu 305 310 315 320 Gly Thr Leu Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro                 325 330 335 Gly Glu Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val             340 345 350 Ser Gln Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala         355 360 365 Ala Met Met Leu Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg     370 375 380 Gln Arg Leu Ile His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp 385 390 395 400 Phe Pro Glu Asp Gln Arg Val Leu Thr Pro Asn Leu Val Ala Leu                 405 410 415 Pro Pro Ser Thr His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val             420 425 430 Trp Ser Ala His Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg         435 440 445 Cys Ala Pro Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser     450 455 460 Gly Lys Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val 465 470 475 480 Cys Arg Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala                 485 490 495 Arg Cys Cys Leu Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro             500 505 510 Pro Ala Glu Ala Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly         515 520 525 His Val Leu Thr Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly     530 535 540 Thr His Lys Pro Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys 545 550 555 560 Val Gly His Arg Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro                 565 570 575 Gly Leu Glu Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu             580 585 590 Gln Val Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser         595 600 605 Ala Leu Pro Gly Thr Ser Val Val Leu Gly Ala Tyr Ala Val Asp Asn     610 615 620 Thr Cys Val Val Arg Ser Ser Asp Val Ser Thr Thr Gly Ser Thr Ser 625 630 635 640 Glu Gly Ala Val Thr Ala Val Ala Ile Cys Cys Arg Ser Ser His His                 645 650 655 Ala Gln Ala Ser Gln Glu Leu Gln Asp Val His His His His His             660 665 670 <210> 14 <211> 22 <212> PRT <213> Artificial <220> <223> Synthetic <400> 14 Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp 1 5 10 15 Gly Gly Ser Leu Val Glu             20 <210> 15 <211> 16 <212> PRT <213> Artificial <220> <223> Synthetic <400> 15 Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly Gly Ser Leu Val Glu 1 5 10 15 <210> 16 <211> 12 <212> PRT <213> Artificial <220> <223> Synthetic <400> 16 Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro 1 5 10 <210> 17 <211> 376 <212> PRT <213> Artificial <220> <223> Synthetic <400> 17 Gln Glu Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg 1 5 10 15 Ser Glu Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala             20 25 30 Thr Phe His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr         35 40 45 Val Val Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr     50 55 60 Ala Arg Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys 65 70 75 80 Ile Leu His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met                 85 90 95 Ser Gly Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His His Val Asp Tyr             100 105 110 Ile Glu Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu         115 120 125 Glu Arg Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro     130 135 140 Asp Gly Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln 145 150 155 160 Ser Asp His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu                 165 170 175 Asn Val Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys             180 185 190 Cys Asp Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp         195 200 205 Ala Gly Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn     210 215 220 Cys Gln Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe 225 230 235 240 Ile Arg Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu                 245 250 255 Leu Pro Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln             260 265 270 Arg Leu Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe         275 280 285 Arg Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile     290 295 300 Thr Val Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr 305 310 315 320 Leu Gly Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu                 325 330 335 Asp Ile Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln             340 345 350 Ser Gly Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met         355 360 365 Met Leu Ser Ala Glu Pro Glu Leu     370 375

Claims (10)

Wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises the CDRs LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 The amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5 , And the amino acid sequence of LCDR3 is represented by SEQ ID NO: 6. 6. An antibody or an antigen-binding fragment thereof, which binds to human PCSK9. The antibody of claim 1, wherein the heavy chain variable region (HCVR) and the light chain variable region (LCVR), wherein the amino acid sequence of HCVR is represented by SEQ ID NO: 7 and the amino acid sequence of LCVR is represented by SEQ ID NO: 8, Antigen-binding fragment. 3. The antibody of claim 2 comprising two HCVRs and two LCVRs wherein the amino acid sequence of each HCVR is represented by SEQ ID NO: 7 and the amino acid sequence of each LCVR is represented by SEQ ID NO: 8, or an antigen- . 2. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises an immunoglobulin heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is represented by SEQ ID NO: 9 and the amino acid sequence of LC is represented by SEQ ID NO: . The antibody of claim 4, comprising two heavy chains (HC) and two light chains (LC), wherein the amino acid sequence of each HC is represented by SEQ ID NO: 9 and the amino acid sequence of each LC is represented by SEQ ID NO: 10 Or an antigen-binding fragment thereof. (HC) and two light chains (LC), wherein the amino acid sequence of each HC is represented by SEQ ID NO: 9, and the amino acid sequence of each LC is represented by SEQ ID NO: 10. Wherein the heavy chain consists of two heavy chains and two light chains, wherein the amino acid sequence of each heavy chain is represented by SEQ ID NO: 9 and the amino acid sequence of each light chain is represented by SEQ ID NO: 10. A pharmaceutical composition for treating hyperlipidemia or hypercholesterolemia, comprising the antibody of claim 1 or an antigen-binding fragment thereof and at least one pharmaceutically acceptable carrier, diluent or excipient. A pharmaceutical composition for treating hyperlipidemia or hypercholesterolemia in a patient in need of treatment comprising an effective amount of the antibody of claim 1 or an antigen-binding fragment thereof. A pharmaceutical composition for treating hyperlipidemia or hypercholesterolemia in a patient in need of treatment comprising an effective amount of the antibody of claim 6.